Gene amplification plays a pivotal role in human malignancy. Highly amplified genes frequently localize to extrachromosomal double minutes (dmin), which usually segregate to daughter cells in association with mitotic chromosomes. We and others had shown that treatment with low-dose hydroxyurea (HU) results in the elimination of dmin and reversion of the cancer cell phenotype. HU treatment in early S-phase, when dmin are replicated, results in their detachment from chromosomes at the next M-phase, leading to the appearance of micronuclei enriched in dmin, followed by their elimination. In this article, we examined the effect of low-dose HU on the behavior of dmin in relation to DNA damage induction by simultaneously monitoring LacO-tagged dmin and phosphorylated histone H2AX (gammaH2AX). As expected, treatment with low-dose HU induced numerous gammaH2AX foci throughout the nucleus in early S-phase, and these rarely coincided with dmin. Most chromosomal gammaH2AX foci disappeared by metaphase, whereas, unexpectedly, those that persisted frequently associated with dmin. We found that these dmin aggregated, detached from anaphase chromosomes, and apparently formed micronuclei. Because gammaH2AX foci likely represent DNA double strand breaks (DSBs), the response to DSBs sustained by extrachromosomal dmin appears to be different from that sustained by chromosomal loci, which may explain why DSB-inducing agents cause the selective elimination of dmin.
Copyright (c) 2007 Wiley-Liss, Inc.